1. Field of the Invention
The present invention is directed towards methods and compositions of amphiphathic compounds used as drugs to directly regulate G protein function in vivo. More particularly, the present invention is directed towards amphipathic compounds based on the key structural determinants of modified mastoparan and receptor-derived peptides. Such compounds would be used as novel drugs to combat a variety of disease states in which G proteins are intimately involved, e.g., asthma, gastric ulcers, cardiovascular disease, allergies, Parkinson's disease, small cell carcinoma of the lung, and the like.
2. Description of the Related Art
G proteins (guanine nucleotide binding regulatory proteins) are important to regulatory mechanisms operating in all human cells. Impairment of their function can perturb the cell's response to hormonal signals and adversely affect many intracellular metabolic pathways, thus contributing to the development and maintenance of a wide variety of disease states.
When functioning normally, G proteins act as an integral part of the signal transducing mechanism by which extracellular hormones and neurotransmitters convey their signals through the plasma membrane of the cell and thus elicit appropriate intracellular responses.
In its simplest terms, the signal transducing mechanism can be said to comprise three distinct components. A receptor protein with an extracellular binding site specific for a given agonist, such as the .beta.-adrenergic receptor; a membrane-bound effector protein that when activated catalyzes the formation or facilitates the transport of an intracellular second messenger, an example is adenylate cyclase which produces cyclic AMP (cAMP); and a third protein which functions as a communicator between these two. G proteins fulfill this vital role as communicator in the generation of intracellular responses to extracellular hormones and agonists.
G proteins are composed of three polypeptide subunits, namely G.alpha., G.beta. and G.gamma.. The conformation of each subunit and their degree of association changes during the signal transducing mechanism. These changes are associated with the hydrolysis of the nucleotide GTP to form GDP and P.sub.i (GTPase activity). The binding sites for GTP, GDP and the GTPase catalytic site reside in the .alpha. subunit.
The G protein cycle which occurs each time a signal is conveyed across the membrane can be summarized as follows:
In an unstimulated cell the G proteins are found in the resting state in which .alpha., .beta. and .gamma. are complexed together and GDP is bound to G.alpha.. The binding of an appropriate hormone or agonist to the receptor changes its conformation and causes it to activate the G protein by displacing GDP and allowing GTP to bind. This is the rate-limiting step of the G protein cycle. When GTP is bound to G.alpha. it may dissociate from .beta..gamma. and is able to bind to, and activate, adenylate cyclase which releases cAMP into the cytoplasm. GTP is then hydrolysed to GDP and the cycle is complete.
The series of complex interactions has evolved to allow signal amplification, such that a single hormone-receptor complex can trigger the production of several hundred second messenger molecules, such as cAMP. cAMP is a potent second messenger that binds to and activates protein kinase A (PKA). PKA was first shown to play a role in glycogen metabolism and is now known to influence a variety of processes including transcription.
A further attribute inherent in this system is that it allows several different receptors to interact with a signal-generating enzyme. Some act in such a way to activate the enzyme and some to inhibit it. This involves distinct .alpha. subunits G.sub.s.alpha. (stimulatory) and G.sub.i.alpha. (inhibitory) that combine with the same .beta..gamma. complex to form stimulatory or inhibitory G proteins. An example of a receptor that interacts with G.sub.i to lower the concentration of cAMP is the .alpha..sub.2 -adrenergic receptor. The integration of the signals from G.sub.s and G.sub.i is one of the ways in which the level of cAMP in the cell can be fine-tuned in response to several different extracellular agonists.
Although G proteins were first identified and characterized in relation to the adenylate cyclase system, as discussed above, it is now apparent that they are involved in many other aspects of cell signalling. In particular, certain G proteins act in the signal transducing pathways that activate phospholipase C. This is a key enzyme that catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP.sub.2) to form diacylglycerol (DG) and inositol 1,4,5-triphosphate (IP.sub.3). DG causes the activation of protein kinase C (PKC) which phosphorylates a certain sub-set of cellular proteins and modulates their activity. For example, PKC is important in controlling the intracellular pH and in the transcriptional activation of specific genes. IP.sub.3 is a small water-soluble molecule that causes the release of Ca.sup.2+ from intracellular stores where it has been sequestered. CA.sup.2+ itself is a potent intracellular messenger that plays a vital role in several metabolic and homeostatic pathways.
As has been shown, the importance of G proteins to the well-being of the cell cannot be stressed too much. It is not therefore surprising that any modulation of G protein function can have catastrophic consequences. Such is the case in individuals who are genetically deficient in G.sub.s, their decreased responses to many hormones cause impaired growth, mental retardation and severe metabolic abnormalities.
Cholera and pertussis toxins also exert their effects through G proteins. Cholera toxin catalyzes the irreversible modification of G.sub.s.alpha., by ADP-ribosylation, which destroys its GTPase activity and locks it into an active state. The resulting prolonged elevation in cAMP levels within the intestinal epithelial cells causes a large efflux of Na.sup.+ and water into the gut, which can prove to be fatal. Pertussis toxin, made by the bacterium that produces whooping cough, alters the G.sub.i.alpha. protein in a similar manner and prevents the inhibition of adenylate cyclase, thus also raising cAMP levels.
Following the identification of G proteins as important elements in many pathological conditions, several attempts have been made to design effective treatment strategies. However, each particular method employed suffers from certain drawbacks.
Many drugs are currently directed towards the hormone receptors themselves, such as the .beta.-adrenergic agents used in the treatment of asthma. The usefulness of this class of drugs is limited by the problems of receptor desensitization and down regulation. In the normal physiological state the amount of functional receptor on a cell's surface is not constant, but is modulated in response to the hormone level. Down regulation of receptors is a general response to a high level of circulating hormone or agonist. The reduction of functional cell surface receptors desensitizes the cell and higher concentrations of agonist do not elicit an appropriately higher response. Any therapeutic agent which involves binding to the receptor is therefore partly flawed by the reduction in the number of receptors which will subsequently occur. It is evident that a downward spiral can result in which ever increasing doses are required to obtain the same effect, and at each dose the number of effective receptors would decline further.
The present invention seeks to by-pass the problem of receptor down regulation by using novel compounds that directly regulate G protein function.
Mastoparan (MP) is a peptide toxin from wasp venom that has been shown to directly stimulate G protein activation. MP is the prototype of a family of peptide toxins, collectively known as mastoparans, that form amphiphilic .alpha. helices. MP has been shown to stimulate guanine nucleotide exchange by G proteins in a manner similar to that of G protein-coupled receptors.
When MP is bound to a phospholipid bilayer, it forms an .alpha. helix that lies parallel to the plane of the membrane, with its hydrophobic face within the bilayer and its four positive charges (3 lysyl residues and terminal amino group) facing outward. G protein-coupled receptors are also believed to display clusters of positive charge near the inner surface of the membrane, some of which are predicted to form amphiphilic helices.
These observations gave rise to the idea that mastoparans could be used to directly regulate G protein function in vivo, and so form the basis of a novel family of drugs that would not suffer from the drawbacks of receptor desensitization. Such G protein-targeted drugs could also be used in cases where G protein mediated responses are important but in which no manipulatable receptor input is available.
This invention relates to the development of the above idea and the intelligent modification of MP to engineer features providing the optimum activity and desired G protein specificity.